Journal: The Journal of Neuroscience

Article Title: Semaphorin-5B Controls Spiral Ganglion Neuron Branch Refinement during Development

doi: 10.1523/JNEUROSCI.0113-19.2019

Figure Lengend Snippet: Primary antibodies and tools/software used in this study

Article Snippet: Antibodies used in this study include the following ( ): mouse-anti-Tuj1 (Covance, MMS-435P; 1:500), rabbit-anti-GAP43 (Millipore, AB5220; 1:1000), rabbit-anti-dsRed (Clontech, 632496; 1:2000), rabbit-anti-MyosinVI (MyoVI; Proteus Biosciences, 25-6791; 1:1000), goat-anti-Sox2 (R&D Systems, AF2018; 1:400), goat-anti-MyoVI ( Coate et al., 2015 ), goat-anti-PlexinA1 (R&D Systems, AF4309; 1:100), goat-anti-PlexinA3 (R&D Systems, AF4075; 1:100), and chicken-anti-neurofilament (NF200; Aves Labs, NFH; 1:2000). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Product Category Source/vendor (catalog #) RRID Mouse α-Tuj1 (TUBB3) Antibody BioLegend (801213) AB_2728521 Rabbit α-Gap43 Antibody Millipore (AB5220) AB_2107282 Rabbit α-dsRed Antibody Takara Bio (632496) AB_10013483 Rabbit α-Myo6 Antibody Proteus Biosciences (25-6791) AB_10013626 Goat α-Sox2 Antibody R&D Systems (AF2018) AB_355110 Goat α-Myo6 Antibody Section on Developmental Neuroscience; NIDCD AB_2783873 Goat α-PlxnA1 Antibody R&D Systems (AF4309) AB_10645644 Goat α-PlxnA3 Antibody R&D Systems (AF4075) AB_2166395 Chicken α-NF200 Antibody Aves Labs (NFH) AB_2313552 Human IgG-Fc protein Antibody Jackson ImmunoResearch Labs (009-000-008) AB_2337046 Bitplane Imaris Tools/Software Oxford Instruments SCR_007370 GraphPad Prism Tools/Software GraphPad SCR_002798 Fiji Tools/Software ImageJ SCR_002285 Open in a separate window Primary antibodies and tools/software used in this study

Techniques:

Journal:

Article Title: Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells *

doi: 10.1074/jbc.M800109200

Figure Lengend Snippet: Detection of HA-mediated Nanog nuclear localization in MCF-7 and SK-OV-3.ipl cells. I, nuclear fraction isolated from MCF-7 (I, panel A) or SK-OV-3.ipl cells (I, panel B) (untreated (lane 1) or treated with HA (50 μg/ml) for 30 min (lane 2) or pretreated with anti-CD44 for 1 h followed by 30 min of HA (50 μg/ml) treatment (lane 3) or pretreated with NanogsiRNA followed by 30 min of HA treatment (lane 4) or pretreated with scrambled sequence siRNA followed by 30 min of HA treatment (lane 5) or transfected with NanogcDNA (lane 6)) were immunoblotted with anti-Nanog antibody (a) or anti-lamin A/C antibody (b) (as a loading control). II, detection of Nanog target gene expression in both MCF-7 (panel A) and SK-OV-3.ipl cells (panel B). The expression of Nanog target genes (e.g. Rex1 and Sox2) was measured using specific primers and Q-PCR in tumor cells according to the procedures described under “Materials and Methods.” Total RNA isolated from either MCF-7 (panel A) or SK-OV-3.ipl cells (panel B) (untreated (lane 1) or treated with HA (50 μg/ml) for 24 h (lane 2) or pretreated with anti-CD44 for 1 h followed by 24 h HA (50μg/ml) treatment (lane 3) or pretreated with NanogsiRNA followed by 24 h HA treatment (lane 4) or pretreated with scrambled sequence siRNA followed by 24 h HA treatment (lane 5) or transfected with NanogcDNA (lane 6)) was reverse-transcribed and subjected to Q-PCR using Rex1 (panel A, a and panel B, a)-specific or Sox2 (panel A, b and panel B, b)-specific primer pairs as described under “Materials and Methods.” Relative mRNA expression levels of Rex1 or Sox2 in various treatments were calculated after normalization with 36B4 mRNA levels as determined by Q-PCR. The values expressed in this figure represent an average of triplicate determinations of three experiments with a standard deviation less than ±5%.

Article Snippet: Both sheep anti-Rex1 antibody and goat anti-Sox2 antibody were obtained from R & D Systems (Minneapolis, MN).

Techniques: Isolation, Sequencing, Transfection, Targeted Gene Expression, Expressing, Reverse Transcription, Standard Deviation

Journal:

Article Title: Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells *

doi: 10.1074/jbc.M800109200

Figure Lengend Snippet: A proposed model for CD44 interaction with Nanog signaling (I) and ankyrin (II) during HA-activated multidrug resistance and tumor progression. I, upon binding of HA, CD44 is first tightly coupled with Nanog in a complex (step 1) followed by an increase of Nanog in the nucleus. Nanog then causes transcriptional activation (step 2a) and the expression of its target genes such as Rex1 and Sox2 (step 3a). Some Nanog also forms a complex with Stat-3 in the nucleus and induces Stat-3-specific transcriptional activation (step 2b) leading to tumor cell growth and MDR-1 gene expression (step 3b) (and localization at the plasma membrane) (step 4); II, HA binding also promotes ankyrin-MDR1 (P-gp) association with CD44 (step A). This complex formation results in an efflux of chemotherapeutic drugs (step B). The coordinated HA-mediated CD44 activation of Nanog/Nanog-Stat-3 signaling (I) and the ankyrin-based cytoskeleton (II) is proposed as a possible mechanism underlying various tumor stem cell-specific behaviors, including transcriptional activation, tumor cell growth, and multidrug resistance during both breast and ovarian tumor progression.

Article Snippet: Both sheep anti-Rex1 antibody and goat anti-Sox2 antibody were obtained from R & D Systems (Minneapolis, MN).

Techniques: Binding Assay, Activation Assay, Expressing, Membrane

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A and B) Total distance and velocity of Barnes maze test of (A) mGfap-Cre:Sox2 fl/fl (n = 9 control, 11 Sox2 cKO) and (B) Aldh1l1-CreER T2 :Sox2 fl/fl (n = 11 control, 11 Sox2 icKO) mice. (C–F) Movement tracing and quantification of open field test of (C and D) mGfap-Cre:Sox2 fl/fl (n = 13 control, 17 Sox2 cKO) and (E and F) Aldh1l1-CreER T2 :Sox2 fl/fl (n = 12 control, 10 Sox2 icKO) mice. (G and H) Movement tracing and quantification of home-cages test. n = 12 control, 10 Sox2 icKO. (I) Diagram showing cortical (Ctx) neuronal projections onto dorsal striatal (Str) medium spinal neurons (MSNs) in the corticostriatal circuitry. (J and K) Representative images and quantification of VGlut1 and PSD95 in the dorsal striatum. Arrowheads, VGlut1/PSD95 co-labeled puncta. n = 4 control, 4 Sox2 icKO. (L and M) Current tracing and quantification of miniature excitatory postsynaptic currents (mEPSCs) of dorsal Str MSNs (mEPSC frequency, n = 11 control, 11 Sox2 icKO; mEPSC amplitude, n = 8 control, 8 Sox2 icKO) and Ctx neurons (mEPSC frequency, n = 10 control, 10 Sox2 icKO; mEPSC amplitude, n = 12 control, 12 Sox2 icKO). (N and O) Representative images and density of C-fos + NeuN + cells in the dorsal striatum. n = 4 control, 5 Sox2 icKO. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics. Animal ages: (A–K) 2 months, (L and M) P35 (tamoxifen P15/P16), and (N and O) P21 (tamoxifen P4–P9). n, biological replicates. Scale bars: (J) 1 μm and (N) 10 μm.

Article Snippet: The digested chromatin was collected and then subjected to IP with SOX2 antibody or normal goat IgG (Cat# AB-108-C, R&D Systems) as control overnight at 4°C with rotation.

Techniques: Control, Labeling, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A) Experimental design for (B)–(H). (B) Representative images of maximal projection and 3D reconstruction (IMARIS) of mG + astrocytes. (C and D) Surface area and enclosed volume of mG + astrocytes. Graphed as means of each astrocyte in Aldh1l1-CreER T2 :Sox2 fl/fl mice (left, n = 28 control, 43 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (E–H) Automatic tracing of mG + astrocyte processes by the filament tool of IMARIS and quantifications. Graphed as means of each astrocyte (left, n = 19 control, 17 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (I) Left, experimental design for (J)–(P); right, immunofluorescence of SOX2 and GFAP and quantification. n = 4 control, 4 Sox2 icKO. (J) Ctx astrocytes visualized by SR101 (arrows) for whole-cell recordings. (K) Voltage steps for astrocyte recording, from −180 to +20 mV with a step size of 10 mV. (L and M) Representative current tracing (L) and I-V curve (M) of one Sox2 -deficient and one Sox2 -intact astrocyte. (N1–N3) Quantification of cell capacitance (N1, n = 23 control, 18 Sox2 icKO), input resistance (N2, n = 20 control, 19 Sox2 icKO), and resting membrane potential (N3, n = 20 control, 17 Sox2 icKO) of Ctx astrocytes. (O) Western blot and quantification of Kir4.1 in the brain. n = 4 control, 4 Sox2 icKO. (P) Representative images and quantification of Kir4.1 intensity in the cortex. n = 4 control, 4 Sox2 icKO. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics. n, biological replicates. Scale bars, (B, E, I, J, and P) 20 μm.

Article Snippet: The digested chromatin was collected and then subjected to IP with SOX2 antibody or normal goat IgG (Cat# AB-108-C, R&D Systems) as control overnight at 4°C with rotation.

Techniques: Control, Immunofluorescence, Membrane, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A) Genomic distribution of SOX2-bound peaks. (B) Top enriched motif CAAAG is present in 99.7% of the top 1,059 SOX2-bound sites . (C–E) Significantly enriched GO terms among the top 1,059 SOX2-bound genomic regions. (F) Overrepresented Disease Ontology terms of the top 1,059 SOX2-bound genomic regions. (G) Genome browser views of SOX2 binding and H3K27Ac overlapping in the astrocytic genes Gfap, Apq4, Wnt7a, Ntrk2, Kcnj10 , and Sparcl1 . (H) ChIP-qPCR verification of SOX2 binding at the sites marked by stars in (G) in primary astrocytes cultured in serum-free medium. n = 3 biological replicates. (I) Representative downregulated genes identified by bulk RNA-seq. Asterisks indicate SOX2-bound genes . (J) Western blot assay of representative downregulated DEGs in the brain of m Gfap-Cre:Sox2 fl/fl mice. n = 3 control, 3 Sox2 cKO. (K) qRT-PCR assays for representative SOX2-bound genes in primary astrocytes. n = 4 control, 4 Sox2 cKO. n, biological replicates. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics.

Article Snippet: The digested chromatin was collected and then subjected to IP with SOX2 antibody or normal goat IgG (Cat# AB-108-C, R&D Systems) as control overnight at 4°C with rotation.

Techniques: Binding Assay, ChIP-qPCR, Cell Culture, RNA Sequencing, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A and B) Genome browser view of SOX2-bound sites and co-occupancy by H3K27Ac in in Slc1a2 and Slc1a3 genes. (C) ChIP-qPCR verification of SOX2 binding to Slc1a2 in primary astrocytes. n = 3 biological replicates. (D) Representative images and quantification of SOX2/GFAP in the cortex of Aldh1l1-CreR T2 :Sox2 fl/fl mice. n = 4 control, 4 Sox2 icKO. (E and F) Western blot images and quantification of GLT-1 in the brain. 62 (monomer), 125 (dimer), and 250 kD (tetramer) of GLT-1. n = 4 control, 4 Sox2 icKO. (G and H) Representative DAB histological images and quantification of GLT1 in the brain. n = 3 control, 3 Sox2 icKO. (I) Representative immunofluorescence of GFAP and SOX2 in serum-free primary astrocytes (14 days in vitro [DIV14]) isolated from mGfap-Cre:Sox2 fl/fl mice. (J) qRT-PCR assay for Slc1a2 and Slc1a3 in primary astrocytes. n = 4 control, 4 Sox2 cKO. (K) Glutamate uptake of primary astrocytes at 1.5 and 4 h after glutamate incubation. n = 6 control, 6 Sox2 cKO. (D–H) P21 Aldh1l1-CreR T2 :Sox2 fl/fl mice (tamoxifen injection at P4–P9). n, biological replicates. Scale bars, (D) 20 μm, (G) 200 μm, and (I) 50 μm.

Article Snippet: The digested chromatin was collected and then subjected to IP with SOX2 antibody or normal goat IgG (Cat# AB-108-C, R&D Systems) as control overnight at 4°C with rotation.

Techniques: ChIP-qPCR, Binding Assay, Control, Western Blot, Immunofluorescence, In Vitro, Isolation, Quantitative RT-PCR, Incubation, Injection

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The digested chromatin was collected and then subjected to IP with SOX2 antibody or normal goat IgG (Cat# AB-108-C, R&D Systems) as control overnight at 4°C with rotation.

Techniques: Control, Protein Extraction, Recombinant, Electron Microscopy, Injection, Magnetic Cell Separation, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Glutamate Assay, Labeling, SYBR Green Assay, Chromatin Immunoprecipitation, Software, Activity Assay

Journal: eLife

Article Title: Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex

doi: 10.7554/eLife.32332

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Sox2 (goat polyclonal) , R + D Systems , R and D Systems Cat# AF2018, RRID: AB_355110 , (1:500).

Techniques: Recombinant, Amplification, Clone Assay, Sequencing, Software